Immunophenotype profile by flow cytometry reveals different subtypes of extracellular vesicles in porcine seminal plasma

Cell Communication and Signaling, Год журнала: 2024, Номер 22(1)

Опубликована: Янв. 23, 2024

Abstract Background Porcine seminal plasma (SP) is endowed with a heterogeneous population of extracellular vesicles (sEVs). This study evaluated the immunophenotypic profile by high-sensitivity flow cytometry eight sEV subpopulations isolated according to their size (small [S-sEVs] and large [L-sEVs]) from four different SP sources, namely three ejaculate fractions (the first 10 mL sperm rich fraction [SRF-P1], remaining SRF [SRF-P2], post-SRF [PSRF]) entire (EE). Methods Seminal EVs were using exclusion chromatography-based protocol six pools (five ejaculates/pool) each source characterized complementary approaches including total protein (BCA™assay), particle distribution (dynamic light scattering), morphology (transmission electron microscopy), purity (albumin Western blot). Expression CD9, CD63, CD81, CD44 HSP90β was analyzed in all MIFlowCyt-EV guidelines, an accurate calibration, controls, discrimination CFSE-labelling. Results Each subpopulation exhibited specific profile. The percentage sEVs positive for CD81 differed between S- L-sEVs ( P < 0.0001). Specifically, CD9 CD63 higher that lower than sources. However, HSP90β-positive S-sEVs SRF-P1 EE samples. also among sources 0.0001), being highest PSRF Notably, virtually expressed (range: 88.04–98.50%). Conclusions demonstrated utility immunophenotyping, allowing identification distinct may have cellular origin, cargo, functions, target cells.

Язык: Английский

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Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism

Theriogenology, Год журнала: 2024, Номер 219, С. 167 - 179

Опубликована: Фев. 26, 2024

Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using size-exclusion chromatography-based procedure characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity EV-protein markers (flow cytometry; albumin, CD81, HSP90β). characterization confirmed existence subsets high (low albumin content) differed (S- L-sEVs). In was performed matured oocytes frozen-thawed spermatozoa IVF medium supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO2 humidified atmosphere) three different concentrations each subset: 0 (control, without sEVs), 0.1, 0.2 mg/mL. first experiment showed sEVs, regardless subset concentration, decreased penetration rates total efficiency (P < 0.0001). subsequent experiment, it shown impaired ability to bind zona pellucida following subset, bound sperm but not oocytes, indicating would affect functionality oocyte functionality. lack by incubating prior IVF, achieving sperm-zona binding results similar those control. last conducted under conditions, analyzed terms tyrosine phosphorylation, acrosome integrity metabolism. did phosphorylation or integrity, influence metabolism decreasing ATP production capacitating conditions. conclusion, this demonstrated presence impairs outcomes, most likely altering

Язык: Английский

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Differential protein repertoires related to sperm function identified in extracellular vesicles (EVs) in seminal plasma of distinct fertility buffalo (Bubalus bubalis) bulls

Frontiers in Cell and Developmental Biology, Год журнала: 2024, Номер 12

Опубликована: Июль 29, 2024

Buffalo bulls are backbone of Indian dairy industry, and the quality semen donating determine overall production efficiency farms. Seminal plasma harbor millions lipid bilayer nanovesicles known as extracellular vesicles (EVs). These EVs carry a heterogenous cargo essential biomolecules including fertility-associated proteins which contribute to fertilizing potential spermatozoa. In this study, we explored size, concentration, complete proteome profiles SP from two distinct fertility groups uncover influencing bull fertility. Through Dynamic Light Scattering (DLS) it was found that purified were present in 7-14 size exclusion chromatographic (SEC) fractions with sizes ranging 146.5 258.7 nm high fertile (HF) low (LF) bulls. Nanoparticle Tracking Analysis (NTA) confirmed seminal up 200 nm, concentrations varying 2.84 6.82 × 10

Язык: Английский

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Reproductive physiology of the boar: What defines the potential fertility of an ejaculate?

Animal Reproduction Science, Год журнала: 2024, Номер 269, С. 107476 - 107476

Опубликована: Апрель 18, 2024

Despite decades of research and handling semen for use in artificial insemination (AI) other assisted reproductive technologies, 5-10% selected boar sires are still considered sub-fertile, escaping current assessment methods sperm quality resilience to preservation. As end-product, the ejaculate (emitted spermatozoa sequentially exposed composite seminal plasma, SP) ought define homeostasis testes, epididymis, accessory sexual glands. Yet, linking findings production biology fertility is suboptimal. The present essay critically reviews how a fertile can help us diagnose both health handling, focusing on -available under development- identify suitable biomarkers cryotolerance fertility. Bulk SP, proteins microRNAs (miRNAs) have, albeit linked function after AI, failed enhance outcomes at commercial level, perhaps just being components complex functional pathway. Hence, focus now interaction sperm-SP, comparing vivo with ex vivo, regarding nano-sized lipid bilayer extracellular vesicles (sEVs) as priority. sEVs transport fragile molecules (lipids, proteins, nucleic acids) which, shielded from degradation, mediate cell-to-cell communication female internal genital tract. Such modulates essential processes, immunological tolerance. be harvested, characterized, stored, manipulated, e.g. used andrological diagnosis, selection breeders, alternatively additives improve cryosurvival

Язык: Английский

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Small and Large Extracellular Vesicles of Porcine Seminal Plasma Differ in Lipid Profile

International Journal of Molecular Sciences, Год журнала: 2024, Номер 25(13), С. 7492 - 7492

Опубликована: Июль 8, 2024

Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs) that remains poorly characterized. This study aimed to characterize the lipidomic profile two subsets differently sized sEVs, small (S-) and large (L-), isolated from porcine seminal by size-exclusion chromatography characterized an orthogonal approach. High-performance liquid chromatography-high-resolution mass spectrometry was used for analysis. A total 157 lipid species 14 classes 4 major categories (sphingolipids, glycerophospholipids, glycerolipids, sterols) were identified. Qualitative differences limited cholesteryl ester present only in S-sEVs. L-sEVs had higher levels all quantified due their larger membrane surface area. The distribution pattern different, especially sphingomyelins (more S-sEVs) ceramides L-sEVs). In conclusion, this reveals suggesting they differ biogenesis functionality.

Язык: Английский

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Cryogenic electron microscopy reveals morphologically distinct subtypes of extracellular vesicles among porcine ejaculate fractions

Scientific Reports, Год журнала: 2024, Номер 14(1)

Опубликована: Июль 13, 2024

Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially livestock species. To better understand their functional role both spermatozoa and endometrial epithelial cells, proper characterization of EVs an essential step. The objective was to phenotypically characterize porcine seminal (sEVs) using cryogenic electron microscopy (cryo-EM), allows visualization native state. Porcine ejaculates released fractions, each containing SP from different source. This sEVs various male reproductive tissues. Two experiments were performed, the first with entire ejaculate (n:6) second three fractions (n:15): 10 mL sperm-rich fraction (SRF-P1) mainly epididymis, remainder SRF (SRF-P2) prostate, post-SRF vesicles. isolated by size exclusion chromatography 1840 cryo-EM sEV images acquired a Jeol-JEM-2200FS/CR-EM. size, density, complexity, peripheral corona layer measured ImageJ software. experiment showed that structurally morphologically heterogeneous, although most (83.1%) small (less than 200 nm), rounded, electrodense, some have coronal layer. There also larger (16.9%) irregularly shaped, more few common SRF-P1 SRF-P2, indicating they originated epididymis prostate. Large abundant post-SRF, heterogeneous. would be explained diversity organs origin.

Язык: Английский

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Isolation and Characterization of Extracellular Vesicle Subsets in Donkey Seminal Plasma

Опубликована: Янв. 1, 2025

Язык: Английский

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Protective role of extracellular vesicles against oxidative DNA damage

Biological Research, Год журнала: 2025, Номер 58(1)

Опубликована: Март 13, 2025

Abstract Background Oxidative stress, a source of genotoxic damage, is often the underlying mechanism in many functional cell disorders. Extracellular vesicles (EVs) have been shown to be key regulators cellular processes and may involved maintaining redox balance. Herein, we aimed develop method assess effects EVs on DNA oxidation using porcine seminal plasma extracellular (sEVs). Results The technique was set cell-free plasmid avoid bias generated by uptake sEVs sperm cells, employing increasing concentrations hydrogen peroxide (H 2 O ) that generate single-strand breaks (SSBs). Because SSBs contain free 3’-OH end allow extension through quantitative PCR, such -and therefore SYBR intensity- showed proportional amount SSB. In next stage, H co-incubated with two size-differentiated subpopulations (small large) permeabilized non-permeabilized whether intravesicular content (IC) or surface protects from oxidative damage. obtained small reduced incidence ( P < 0.05), whereas large had no impact generation > 0.05). IC protective effect against Conclusions Our results suggest sEVs, including peripheral corona layer, exert function alterations are originated mechanisms. addition, our vitro study opens path detect, localize quantify derived different fluids, elucidating their physiopathological states.

Язык: Английский

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Identification of a Non-Invasive Urinary Exosomal Biomarker for Diabetic Nephropathy Using Data-Independent Acquisition Proteomics

International Journal of Molecular Sciences, Год журнала: 2023, Номер 24(17), С. 13560 - 13560

Опубликована: Сен. 1, 2023

Diabetic nephropathy (DN), as the one of most common complications diabetes, is generally diagnosed based on a longstanding duration, albuminuria, and decreased kidney function. Some patients with comorbidities diabetes other primary renal diseases have similar clinical features to DN, which defined non-diabetic disease (NDRD). It necessary distinguish between DN NDRD, considering they differ in their pathological characteristics, treatment regimes, prognosis. Renal biopsy provides gold standard; however, it difficult for this be conducted all patients. Therefore, discover non-invasive biomarkers that can NDRD. In research, urinary exosomes were isolated from midstream morning urine ultracentrifugation combined 0.22 μm membrane filtration. Data-independent acquisition-based quantitative proteomics used define proteome profile (n = 12) NDRD 15) Type 2 mellitus (T2DM) without damage 9), well healthy people 12). each sample, 3372 ± 722.1 proteins identified average. We 371 exosome significantly differentially expressed patients, bioinformatic analysis revealed them mainly enriched immune metabolic pathways. The use least absolute shrinkage selection operator (LASSO) logistic regression further phytanoyl-CoA dioxygenase domain containing 1 (PHYHD1) differential diagnostic biomarker, efficacy was verified another cohort including eight five seven T2DM nine people. Additionally, concentration above 1.203 μg/L established ELISA method. Furthermore, 19 different selected by using protein-protein interaction network LASSO regression, 13 related indicators could reflect level function hyperglycemic management.

Язык: Английский

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Updating Research on Extracellular Vesicles of the Male Reproductive Tract in Farm Animals: A Systematic Review

Animals, Год журнала: 2024, Номер 14(21), С. 3135 - 3135

Опубликована: Окт. 31, 2024

This systematic review examined research studies on extracellular vesicles (EVs) of the male reproductive tract in livestock species to summarize topics and methodologies used, key findings, future directions. PubMed Scopus were searched for time ranges up 1 September 2024, 1383 articles identified. The application screening eligibility criteria resulted selection 79 focusing EVs livestock. Porcine bovine most studied. A variety EV isolation techniques with ultracentrifugation being common. Characterization was a weak point, only 24.05% characterizing according MISEV guidelines. Inadequate characterization compromises reliability results. results 19 that provided good showed from are phenotypically compositionally heterogeneous. These papers also these would be involved regulation sperm functionality. Research remains scarce, further is needed, which should include appropriate aim find efficient methods isolate them assess their involvement functionality spermatozoa cells female genital tract.

Язык: Английский

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